This study aims to determine if house flies are important transmitters of bacterial disease agents, by examining the fate of bacteria within flies and concurrent expression of antibacterial humoral defenses. Adult house flies (Musca domestica L.) feed and breed on feces and other septic substrates. Due to their synanthropic nature and indiscriminate feeding habits, house flies are involved in the epidemiology of >65 diseases that affect humans world-wide. A single house fly can harbor as many as 100 species of pathogenic bacteria, including those that cause diarrheal illnesses and trachoma. In rural areas of developing countries, hyperendemicity of gastroenteritis among infants and children is attributed to filth fly presence in combination with poor sanitation. Control of filth fly populations subsequently results in a sharp reduction in gastroenteritis cases. The World Health Organization (WHO) has reported that diarrheal illnesses are one of the top causes of morbidity and mortality in humans, resulting in 1.5 million deaths per year. Further, the WHO estimates that >6 million people are currently blind due to trachoma infections, a disease vectored by Musca spp. Due to the impact house flies have human health, a further understanding of the role that flies play in transmitting bacteria is tantamount to vector potential studies in other microbe-vector systems (e.g., malaria- mosquitoes). Although house flies have been implicated as mechanical vectors of bacteria, vector potential would be bolstered if bacteria were harbored within the body (e.g., the alimentary canal), and multiplied prior to excretion. Only a few reports have demonstrated the multiplication, persistence and excretion of some species of pathogenic bacteria from house flies. Our preliminary data show that bacteria could multiply within the house fly crop and were transmissible by regurgitation. However, oral transmissibility was only possible for a short window of time. When bacteria were shuttled to the midgut they could not be regurgitated, leaving fecal transmission as the only possible exit route for bacteria. We found that few, if any, bacteria survived passage through the midgut as most were lysed in fecal pellets. The proposed study will further examine the fate of bacteria in flies. We will use 6 human-pathogenic species of bacteria that are GFP-tagged, and determine their temporal and spatial location within the alimentary canal (crop, midgut, hindgut) over 24 h. We also will enumerate viable bacteria from excreta (vomitus, feces). We hypothesize that destruction of bacteria in the fly midgut is mediated by secreted effector molecules. Ingested bacteria face an onslaught of defense mechanisms in the midgut including physical barriers (peritrophic matrix) and secreted humoral defenses (lysozyme and antimicrobial peptides;AMPs). Since these effector molecules directly lyse bacteria, assessing their spatial expression in the alimentary canal could provide insight into their role in antibacterial epithelial defenses. Preliminary Northern analyses show that flies (1) constitutively express lysozyme irrespective of exposure to bacteria and (2) express AMPs in response to bacteria introduced by both injection and feeding. House flies showed a unique temporal expression repertoire that was tailored to each species of bacteria tested, and has not been described in other diptera. The proposed study will further examine the spatial (tissue specific) and temporal expression of these molecules (lysozyme, AMPs) on both the transcriptional and peptide levels, using qRT-PCR and immunoflourescence microscopy, respectively. The innovative approach of the proposed study is to simultaneously examine the house fly-bacteria interaction from both the perspective of the microbe (i.e., location and survival within the alimentary canal) and the house fly (i.e., expression of antibacterial responses when bacteria are in these locations). The interplay between the timing of house fly defenses and concurrent location/status of bacteria underlies transmission potential. This study will further our knowledge of insect immunity in a unique system, and thus can help contribute to studies of vector-microbe interactions. The vector midgut presents not only a challenging environment for the survival and development of microbes, but also an opportunity for investigators to gather information that could possibly lead to novel targets of control. Thus, these analyses could provide useful information applicable to the field of vector biology and control (e.g., putative genes for use in paratransgenesis) or even reveal novel antimicrobial treatments for humans. PUBLIC HEALTH RELEVANCE Due to their predilection for septic food substrates and synanthropic nature, house flies are involved in the epidemiology of numerous bacterial diseases that affect humans, especially diarrheal illnesses and trachoma. Diarrheal illnesses are one of the top causes of morbidity and mortality in humans, resulting in 1.5 million deaths per year world-wide, especially in infants and young children. The WHO estimates that >6 million people are currently blind due to trachoma infections. Musca spp. are vectors for trachoma, and have long been implicated in maintaining hyperendemicity of the disease in the rural areas of developing countries. Additionally, house flies are reservoirs for antibiotic resistant bacteria, which poses a risk to humans world-wide. Due to the clear impact of house flies on human health, a further understanding of the role that flies play in harboring or transmitting bacteria is tantamount to studies of vector potential in other microbe-vector systems. The results of this study will broadly contribute to a greater understanding of the role of house flies in the epidemiology of pathogenic bacteria that infect humans.